miR-146a-/- mice model reveals that NF-κB inhibition reverts inflammation-driven myelofibrosis-like phenotype.

Emerging evidence shows the crucial role of inflammation (particularly NF-κB pathway) in the development and progression of myelofibrosis (MF), becoming a promising therapeutic target. Furthermore, tailoring treatment with currently available JAK inhibitors (such as ruxolitinib or fedratinib) does not modify the natural history of the disease and has important limitations, including cytopenias. Since recent studies have highlighted the role of miR-146a, a negative regulator of the NF-κB pathway, in the pathogenesis of MF; here we used miR-146a-/- (KO) mice, a MF-like model lacking driver mutations, to investigate whether pharmacological inhibition of JAK/STAT and/or NF-κB pathways may reverse the myelofibrotic phenotype of these mice. Specifically, we tested the JAK1/2 inhibitor, ruxolitinib; the NF-κB inhibitor via IKKα/ß, BMS-345541; both inhibitors in combination; or a dual inhibitor of both pathways (JAK2/IRAK1), pacritinib. Although all treatments decreased spleen size and partially recovered its architecture, only NF-κB inhibition, either using BMS-345541 (alone or in combination) or pacritinib, resulted in a reduction of extramedullary hematopoiesis, bone marrow (BM) fibrosis and osteosclerosis, along with an attenuation of the exacerbated inflammatory state (via IL-1ß and TNFα). However, although dual inhibitor improved anemia and reversed thrombocytopenia, the combined therapy worsened anemia by inducing BM hypoplasia. Both therapeutic options reduced NF-κB and JAK/STAT signaling in a context of JAK2V617F-driven clonal hematopoiesis. Additionally, combined treatment reduced both COL1A1 and IL-6 production in an in vitro model mimicking JAK2-driven fibrosis. In conclusion, NF-κB inhibition reduces, in vitro and in vivo, disease burden and BM fibrosis, which could provide benefits in myelofibrosis patients.

Digital Microscopy Augmented by Artificial Intelligence to Interpret Bone Marrow Samples for Hematological Diseases.

Analysis of bone marrow aspirates (BMAs) is an essential step in the diagnosis of hematological disorders. This analysis is usually performed based on a visual examination of samples under a conventional optical microscope, which involves a labor-intensive process, limited by clinical experience and subject to high observer variability. In this work, we present a comprehensive digital microscopy system that enables BMA analysis for cell type counting and differentiation in an efficient and objective manner. This system not only provides an accessible and simple method to digitize, store, and analyze BMA samples remotely but is also supported by an Artificial Intelligence (AI) pipeline that accelerates the differential cell counting process and reduces interobserver variability. It has been designed to integrate AI algorithms with the daily clinical routine and can be used in any regular hospital workflow.

Impact of viral hepatitis therapy in multiple myeloma and other monoclonal gammopathies linked to hepatitis B or C viruses.

Subsets of multiple myeloma (MM) and monoclonal gammopathies of undetermined significance (MGUS) present with a monoclonal immunoglobulin specific for hepatitis C virus (HCV), thus are presumably HCV-driven, and antiviral treatment can lead to the disappearance of antigen stimulation and improved control of clonal plasma cells. Here we studied the role of hepatitis B virus (HBV) in the pathogenesis of MGUS and MM in 45 HBV-infected patients with monoclonal gammopathy. We analyzed the specificity of recognition of the monoclonal immunoglobulin of these patients and validated the efficacy of antiviral treatment (AVT). For 18 of 45 (40%) HBV-infected patients, the target of the monoclonal immunoglobulin was identified: the most frequent target was HBV (n=11), followed by other infectious pathogens (n=6) and glucosylsphingosine (n=1). Two patients whose monoclonal immunoglobulin targeted HBV (HBx and HBcAg), implying that their gammopathy was HBV-driven, received AVT and the gammopathy did not progress. AVT efficacy was then investigated in a large cohort of HBV-infected MM patients (n=1367) who received or did not receive anti-HBV treatments and compared to a cohort of HCV-infected MM patients (n=1220). AVT significantly improved patient probability of overall survival (P=0.016 for the HBV-positive cohort, P=0.005 for the HCV-positive cohort). Altogether, MGUS and MM disease can be HBV- or HCV-driven in infected patients, and the study demonstrates the importance of AVT in such patients.

Short-Chain Fatty Acid Production by Gut Microbiota Predicts Treatment Response in Multiple Myeloma.

PURPOSE: The gut microbiota plays important roles in health and disease. We questioned whether the gut microbiota and related metabolites are altered in monoclonal gammopathies and evaluated their potential role in multiple myeloma and its response to treatment. EXPERIMENTAL DESIGN: We used 16S rRNA sequencing to characterize and compare the gut microbiota of patients with monoclonal gammopathy of undetermined significance (n = 11), smoldering multiple myeloma (n = 9), newly diagnosed multiple myeloma (n = 11), relapsed/refractory multiple myeloma (n = 6), or with complete remission (n = 9). Short-chain fatty acids (SCFA) were quantified in serum and tested in cell lines. Relevant metabolites were validated in a second cohort of 62 patients. RESULTS: Significant differences in alpha- and beta diversity were present across the groups and both were lower in patients with relapse/refractory disease and higher in patients with complete remission after treatment. Differences were found in the abundance of several microbiota taxa across disease progression and in response to treatment. Bacteria involved in SCFA production, including Prevotella, Blautia, Weissella, and Agathobacter, were more represented in the premalignant or complete remission samples, and patients with higher levels of Agathobacter showed better overall survival. Serum levels of butyrate and propionate decreased across disease progression and butyrate was positively associated with a better response. Both metabolites had antiproliferative effects in multiple myeloma cell lines. CONCLUSIONS: We demonstrate that SCFAs metabolites and the gut microbiota associated with their production might have beneficial effects in disease evolution and response to treatment, underscoring its therapeutic potential and value as a predictor.

NLRP3 inflammasome activation and symptom burden in KRAS-mutated CMML patients is reverted by IL-1 blocking therapy.

Chronic myelomonocytic leukemia (CMML) is frequently associated with mutations in the rat sarcoma gene (RAS), leading to worse prognosis. RAS mutations result in active RAS-GTP proteins, favoring myeloid cell proliferation and survival and inducing the NLRP3 inflammasome together with the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which promote caspase-1 activation and interleukin (IL)-1ß release. Here, we report, in a cohort of CMML patients with mutations in KRAS, a constitutive activation of the NLRP3 inflammasome in monocytes, evidenced by ASC oligomerization and IL-1ß release, as well as a specific inflammatory cytokine signature. Treatment of a CMML patient with a KRASG12D mutation using the IL-1 receptor blocker anakinra inhibits NLRP3 inflammasome activation, reduces monocyte count, and improves the patient's clinical status, enabling a stem cell transplant. This reveals a basal inflammasome activation in RAS-mutated CMML patients and suggests potential therapeutic applications of NLRP3 and IL-1 blockers.

Posttranslational splicing modifications as a key mechanism in cytarabine resistance in acute myeloid leukemia.

Despite the approval of several drugs for AML, cytarabine is still widely used as a therapeutic approach. However, 85% of patients show resistance and only 10% overcome the disease. Using RNA-seq and phosphoproteomics, we show that RNA splicing and serine-arginine-rich (SR) proteins phosphorylation were altered during cytarabine resistance. Moreover, phosphorylation of SR proteins at diagnosis were significantly lower in responder than non-responder patients, pointing to their utility to predict response. These changes correlated with altered transcriptomic profiles of SR protein target genes. Notably, splicing inhibitors were therapeutically effective in treating sensitive and resistant AML cells as monotherapy or combination with other approved drugs. H3B-8800 and venetoclax combination showed the best efficacy in vitro, demonstrating synergistic effects in patient samples and no toxicity in healthy hematopoietic progenitors. Our results establish that RNA splicing inhibition, alone or combined with venetoclax, could be useful for the treatment of newly diagnosed or relapsed/refractory AML.

Deepening Our Understanding of the Factors Affecting Landscape of Myeloproliferative Neoplasms: What Do We Know about Them?

Myeloproliferative neoplasms (MPNs) arise from the uncontrolled proliferation of hematopoietic stem and progenitor cells in bone marrow. As with all tumors, the development of MPNs is a consequence of alterations in malignant cells and their interaction with other extrinsic factors that support and promote tumor progression. Since the discovery of driver mutations, much work has focused on studying and reviewing the genomic features of the disease but has neglected to delve into the important role that many other mechanisms may play. This review discusses the genetic component of MPNs but focuses mainly on some of the most relevant work investigating other non-genetic factors that may be crucial for the disease. The studies summarized here address MPN cell-intrinsic or -extrinsic factors and the interaction between them through transcriptomic, proteomic and microbiota studies, among others.

CNL and aCML should be considered as a single entity based on molecular profiles and outcomes.

Chronic neutrophilic leukemia (CNL) and atypical chronic myeloid leukemia (aCML) are rare myeloid disorders that are challenging with regard to diagnosis and clinical management. To study the similarities and differences between these disorders, we undertook a multicenter international study of one of the largest case series (CNL, n = 24; aCML, n = 37 cases, respectively), focusing on the clinical and mutational profiles (n = 53 with molecular data) of these diseases. We found no differences in clinical presentations or outcomes of both entities. As previously described, both CNL and aCML share a complex mutational profile with mutations in genes involved in epigenetic regulation, splicing, and signaling pathways. Apart from CSF3R, only EZH2 and TET2 were differentially mutated between them. The molecular profiles support the notion of CNL and aCML being a continuum of the same disease that may fit best within the myelodysplastic/myeloproliferative neoplasms. We identified 4 high-risk mutated genes, specifically CEBPA (ß = 2.26, hazard ratio [HR] = 9.54, P = .003), EZH2 (ß = 1.12, HR = 3.062, P = .009), NRAS (ß = 1.29, HR = 3.63, P = .048), and U2AF1 (ß = 1.75, HR = 5.74, P = .013) using multivariate analysis. Our findings underscore the relevance of molecular-risk classification in CNL/aCML as well as the importance of CSF3R mutations in these diseases.

High-Throughput CRISPR Screening in Hematological Neoplasms.

CRISPR is becoming an indispensable tool in biological research, revolutionizing diverse fields of medical research and biotechnology. In the last few years, several CRISPR-based genome-targeting tools have been translated for the study of hematological neoplasms. However, there is a lack of reviews focused on the wide uses of this technology in hematology. Therefore, in this review, we summarize the main CRISPR-based approaches of high throughput screenings applied to this field. Here we explain several libraries and algorithms for analysis of CRISPR screens used in hematology, accompanied by the most relevant databases. Moreover, we focus on (1) the identification of novel modulator genes of drug resistance and efficacy, which could anticipate relapses in patients and (2) new therapeutic targets and synthetic lethal interactions. We also discuss the approaches to uncover novel biomarkers of malignant transformations and immune evasion mechanisms. We explain the current literature in the most common lymphoid and myeloid neoplasms using this tool. Then, we conclude with future directions, highlighting the importance of further gene candidate validation and the integration and harmonization of the data from CRISPR screening approaches.

Myc-Related Mitochondrial Activity as a Novel Target for Multiple Myeloma.

Mitochondria are involved in the development and acquisition of a malignant phenotype in hematological cancers. Recently, their role in the pathogenesis of multiple myeloma (MM) has been suggested to be therapeutically explored. MYC is a master regulator of b-cell malignancies such as multiple myeloma, and its activation is known to deregulate mitochondrial function. We investigated the impact of mitochondrial activity on the distinct entities of the disease and tested the efficacy of the mitochondrial inhibitor, tigecycline, to overcome MM proliferation. COXII expression, COX activity, mitochondrial mass, and mitochondrial membrane potential demonstrated a progressive increase of mitochondrial features as the disease progresses. In vitro and in vivo therapeutic targeting using the mitochondrial inhibitor tigecycline showed promising efficacy and cytotoxicity in monotherapy and combination with the MM frontline treatment bortezomib. Overall, our findings demonstrate how mitochondrial activity emerges in MM transformation and disease progression and the efficacy of therapies targeting these novel vulnerabilities.

Long-Term Human Hematopoietic Stem Cell Culture in Microdroplets.

We previously reported a new approach for micromanipulation and encapsulation of human stem cells using a droplet-based microfluidic device. This approach demonstrated the possibility of encapsulating and culturing difficult-to-preserve primary human hematopoietic stem cells using an engineered double-layered bead composed by an inner layer of alginate and an outer layer of Puramatrix. We also demonstrated the maintenance and expansion of Multiple Myeloma cells in this construction. Here, the presented microfluidic technique is applied to construct a 3D biomimetic model to recapitulate the human hematopoietic stem cell niche using double-layered hydrogel beads cultured in 10% FBS culture medium. In this model, the long-term maintenance of the number of cells and expansion of hHSCS encapsulated in the proposed structures was observed. Additionally, a phenotypic characterization of the human hematopoietic stem cells generated in the presented biomimetic model was performed in order to assess their long-term stemness maintenance. Results indicate that the ex vivo cultured human CD34+ cells from bone marrow were viable, maintained, and expanded over a time span of eight weeks. This novel long-term stem cell culture methodology could represent a novel breakthrough to improve Hematopoietic Progenitor cell Transplant (HPT) as well as a novel tool for further study of the biochemical and biophysical factors influencing stem cell behavior. This technology opens a myriad of new applications as a universal stem cell niche model potentially able to expand other types of cells.

Efficacy of Antiviral Treatment in Hepatitis C Virus (HCV)-Driven Monoclonal Gammopathies Including Myeloma.

Multiple myeloma (MM) remains an incurable plasma cell malignancy. While its origin is enigmatic, an association with infectious pathogens including hepatitis C virus (HCV) has been suggested. Here we report nine patients with monoclonal gammopathy of undetermined significance (MGUS) or MM with previous HCV infection, six of whom received antiviral treatment. We studied the evolution of the gammopathy disease, according to anti-HCV treatment and antigen specificity of purified monoclonal immunoglobulin, determined using the INNO-LIA™ HCV Score assay, dot-blot assays, and a multiplex infectious antigen microarray. The monoclonal immunoglobulin from 6/9 patients reacted against HCV. Four of these patients received antiviral treatment and had a better evolution than untreated patients. Following antiviral treatment, one patient with MM in third relapse achieved complete remission with minimal residual disease negativity. For two patients who did not receive antiviral treatment, disease progressed. For the two patients whose monoclonal immunoglobulin did not react against HCV, antiviral treatment was not effective for MGUS or MM disease. Our results suggest a causal relationship between HCV infection and MGUS and MM progression. When HCV was eliminated, chronic antigen-stimulation disappeared, allowing control of clonal plasma cells. This opens new possibilities of treatment for MGUS and myeloma.

Protein Carbonylation and Lipid Peroxidation in Hematological Malignancies.

Among the different mechanisms involved in oxidative stress, protein carbonylation and lipid peroxidation are both important modifications associated with the pathogenesis of several diseases, including cancer. Hematopoietic cells are particularly vulnerable to oxidative damage, as the excessive production of reactive oxygen species and associated lipid peroxidation suppress self-renewal and induce DNA damage and genomic instability, which can trigger malignancy. A richer understanding of the clinical effects of oxidative stress might improve the prognosis of these diseases and inform therapeutic strategies. The most common protein carbonylation and lipid peroxidation compounds, including hydroxynonenal, malondialdehyde, and advanced oxidation protein products, have been investigated for their potential effect on hematopoietic cells in several studies. In this review, we focus on the most important protein carbonylation and lipid peroxidation biomarkers in hematological malignancies, their role in disease development, and potential treatment implications.

MEK inhibition enhances the response to tyrosine kinase inhibitors in acute myeloid leukemia.

FMS-like tyrosine kinase 3 (FLT3) is a key driver of acute myeloid leukemia (AML). Several tyrosine kinase inhibitors (TKIs) targeting FLT3 have been evaluated clinically, but their effects are limited when used in monotherapy due to the emergence of drug-resistance. Thus, a better understanding of drug-resistance pathways could be a good strategy to explore and evaluate new combinational therapies for AML. Here, we used phosphoproteomics to identify differentially-phosphorylated proteins in patients with AML and TKI resistance. We then studied resistance mechanisms in vitro and evaluated the efficacy and safety of rational combinational therapy in vitro, ex vivo and in vivo in mice. Proteomic and immunohistochemical studies showed the sustained activation of ERK1/2 in bone marrow samples of patients with AML after developing resistance to FLT3 inhibitors, which was identified as a common resistance pathway. We examined the concomitant inhibition of MEK-ERK1/2 and FLT3 as a strategy to overcome drug-resistance, finding that the MEK inhibitor trametinib remained potent in TKI-resistant cells and exerted strong synergy when combined with the TKI midostaurin in cells with mutated and wild-type FLT3. Importantly, this combination was not toxic to CD34+ cells from healthy donors, but produced survival improvements in vivo when compared with single therapy groups. Thus, our data point to trametinib plus midostaurin as a potentially beneficial therapy in patients with AML.

Protein Carbonylation in Patients with Myelodysplastic Syndrome: An Opportunity for Deferasirox Therapy.

Control of oxidative stress in the bone marrow (BM) is key for maintaining the interplay between self-renewal, proliferation, and differentiation of hematopoietic cells. Breakdown of this regulation can lead to diseases characterized by BM failure such as the myelodysplastic syndromes (MDS). To better understand the role of oxidative stress in MDS development, we compared protein carbonylation as an indicator of oxidative stress in the BM of patients with MDS and control subjects, and also patients with MDS under treatment with the iron chelator deferasirox (DFX). As expected, differences in the pattern of protein carbonylation were observed in BM samples between MDS patients and controls, with an increase in protein carbonylation in the former. Strikingly, patients under DFX treatment had lower levels of protein carbonylation in BM with respect to untreated patients. Proteomic analysis identified four proteins with high carbonylation levels in MDS BM cells. Finally, as oxidative stress-related signaling pathways can modulate the cell cycle through p53, we analyzed the expression of the p53 target gene p21 in BM cells, finding that it was significantly upregulated in patients with MDS and was significantly downregulated after DFX treatment. Overall, our results suggest that the fine-tuning of oxidative stress levels in the BM of patients with MDS might control malignant progression.

Ruxolitinib in combination with prednisone and nilotinib exhibit synergistic effects in human cells lines and primary cells from myeloproliferative neoplasms.

Ruxolitinib is the front-line non-palliative treatment for myelofibrosis (MF). However, a significant number of patients lose or present suboptimal response, are resistant or have unacceptable toxicity. In an attempt to improve response and avoid the adverse effects of this drug, we evaluated the combination of 17 drugs with ruxolitinib in ex vivo models of peripheral blood mononuclear cells from MF patients and cell lines. We found that the combination ruxolitinib and nilotinib had a synergistic effect against MF cells (ΔEC50 nilotinib, -21.6%). Moreover, the addition of prednisone to combined ruxolitinib/nilotinib improved the synergistic effect in all MF samples studied. We evaluated the molecular mechanisms of combined ruxolitinib/nilotinib/prednisone and observed inhibition of JAK/STAT (STAT5, 69.2+11.8% inhibition) and MAPK (ERK, 29.4+4.5% inhibition) signaling pathways. Furthermore, we found that the triple therapy combination inhibited collagen protein and COL1A1 gene expression in human bone marrow mesenchymal cells. Taken together, we provide evidence that combined ruxolitinib/nilotinib/prednisone is a potential therapy for MF, possibly through the anti-fibrotic effect of nilotinib, the immunomodulatory effect of ruxolitinib and prednisone, and the anti-proliferative effect of ruxolitinib. This combination will be further investigated in a phase Ib/II clinical trial in MF.